Project Summary/Abstract Genetic screens in Drosophila have identified mutations that significantly reduce both night- time and daytime sleep. Genes affected by these mutations are expressed in the blood-brain- barrier-forming subperineurial glia of the fly and alter the morphological and biophysical properties of the barrier. We have discovered novel genetic interactions among some of these mutations: surprisingly, certain mutant combinations restore both sleep and blood-brain-barrier function. We propose further studies that could reveal molecular pathways connecting their gene products and clarify their contributions to sleep and barrier function. We discovered that in wild type Drosophila the blood-brain barrier opens and closes with a rhythm that requires a circadian clock. We have also found that barrier permeability is closely connected to sleep need: sleep deprivation opens the barrier, but rebound sleep closes it. What is being exchanged across the barrier in such a dynamic fashion? Nervous system function is protected by a steep concentration gradient of K+ separating the haemolymph and brain. In our proposed studies we will develop tools to quantify K+ flux across the blood brain barrier with high temporal resolution in living flies. Are episodes of sleep and wakefulness correlated with these ion exchanges? Do such measurements reveal features of wake/sleep behavior that are not evident using standard locomotor activity monitoring? Our studies have also shown that sleep mutants reduce lifespan, but in a fashion that can be reversed by time-controlled access to food. These effects require a circadian clock and we will determine which tissues are responsible for this response and whether lifespan restoration depends on sleep recovery. Chronic exposure to psychogenic stressors can have profound, long-lasting effects on both physical and mental health and is often accompanied by a profound loss of sleep. Chronic social isolation provides a means by which a psychogenic stressor can be easily applied for an extended period, and we observe significant reductions in total sleep, day-time sleep, and night-time sleep in isolated flies when compared to sleep in siblings that are group reared. To search for genetic pathways that might respond to isolation-induced stress and depress sleep, comparative RNAseq assays were performed using Drosophila heads collected from group- reared flies, or from flies stressed through chronic isolation. Among the most highly responding genes are those thought to regulate appetite. These map to a small neuronal circuit which we will further characterize to determine its possible role in isolation-induced stress responses affecting sleep and hunger.